Mycobacteria tuberculosis (Mtb) causes the deadly infectious disease, tuberculosis, which continues to plague the world's population, with an estimated one third of the world infected. CarD and RbpA have recently been identified as direct RNA polymerase (RNAP) binding proteins that are essential regulators of transcription in the pathogen Mtb. CarD is widely distributed among bacteria, including Mycobacterium and Thermus species. Our preliminary results indicate that CarD utilizes a distinct molecular mechanism for regulating transcription. RbpA is a regulator of RNAP found only in the actinomycete family of bacteria, including Streptomyces coelicolor (Sco) and Mtb. Like CarD, RbpA has no similarity to other transcription regulators, is essential for growth in mycobacteria and also positively regulates rRNA transcription. Both CarD and RpbA are unusual transcription regulators that do not function according to the standard paradigm for bacterial transcription activators. We propose a combination of in vitro structural and biochemical approaches, and in vivo approaches to elucidate the molecular mechanisms for transcription regulation by CarD and RbpA. Preliminary results include crystal structures of thermus transcription initiation complexes containing CarD (4 -resolution). We propose to improve on these structural results, use biochemical and biophysical approaches to test hypotheses that arise from these structures as well as to confirm that Mtb CarD functions through the same mechanism as thermus CarD. We have also obtained a 2.2 -resolution crystal structure of Mtb RbpA bound to one of its RNAP targets, the Mtb sA. We propose biochemical and biophysical approaches to test hypotheses that arise from this structure and to map additional interactions with the mycobacterial RNAP. Finally, we propose to crystallize and determine the structure of mycobacterial RNAP transcription initiation complexes.